Project Summary Alcohol has wide-reaching effects on the nervous system. The mechanism of action for alcohol is complex, where alcohol interacts specifically and non-specifically with many targets (e.g. receptors, lipids, extra-cellular matrix, protein function, etc.). However, the fundamental mechanisms that underlie the effects of alcohol on different behaviors are poorly understood. Thus, more research is required to identify the key molecules essential for different alcohol behaviors (sensitivity to sedation, withdrawal, tolerance, and drinking) that may be targeted to yield new treatments. We focus here on the role of the BK channel, a calcium and voltage-gated potassium channel, in behavioral responses to alcohol. The highly conserved BK potassium channel is a direct target of ethanol that might be modified to reduce alcohol behaviors with minimal side effects. Unbiased genetic screens revealed that the BK channel represented by far the most important ethanol target for intoxication in Caenorhabditis elegans. Acute ethanol directly activates the BK channel to depress general neuronal activity and behaviors in worm. The BK channel was subsequently implicated as important in various behavioral responses to alcohol in flies, rodents and humans. New research also suggests that the channel may be targeted in a way to minimize side effects. A novel BK channel mutation has been identified (T352I) that prevents effects of intoxication and alcohol withdrawal in a C. elegans model. This mutation alters a single residue that is conserved in worm, mouse and human BK channels. Patch-clamp recordings confirmed that the human BK T352I channel was insensitive to activation by ethanol, but otherwise had normal conductance, K+ selectivity, and only subtle differences in voltage dependence. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes critical for behavioral responses. These results suggest that knocking in the T352I mutation in rodent models may alter ethanol-dependent behaviors without causing gross behavioral impairments, which would advance our understanding of the role of the BK channel in different ethanol-mediated behaviors. For this proposal, we will determine whether the BK channel represents a major target of ethanol to modify behaviors in mammals. This will be done by performing quantitative analysis of alcohol-related behaviors in our new mouse engineered with the BK T352I mutation via CRISPR/Cas9; this mouse was generated with our collaborators Drs. John Pierce, Gregg Homanics, and William Shawlot. We will test whether the T352I mutation reduces sensitivity to ethanol sedation, withdrawal, tolerance, and drinking. We hypothesize that the reduced sensitivity seen in C. elegans carrying the T352I BK mutation will be recapitulated in more complex but analogous behaviors in mutant mice. These studies have the potential to determine whether the BK channel represents a major contributor across different alcohol behaviors in mammals. They will also help elucidate the molecular role of the BK channel in alcohol withdrawal and its potential as a treatment avenue during withdrawal.